Cell Death Screening Using DAPI Staining: A Method for Rapid Screening of IR Induced Clonogenic Cell Death in Cancerous Cell Lines
Transcript
Exposure to ionizing radiations such as X-rays can cause DNA damage, eventually leading to cell death. To screen the effect of ionizing radiations on cell death profiles, begin by taking a culture dish containing adherent cancer cells placed over the surface of a coverslip.
Irradiate the culture dish with X-rays for the desired duration. This treatment causes DNA damage inside the cells. Next, aspirate the spent media from the culture dish and add a suitable fixative. This solution permeates the cells and locks the cellular components in place during subsequent staining steps.
Discard the fixative solution. Remove the coverslip containing X-ray treated cells from the culture dish. Flip the coverslip and place it onto the surface of a glass slide carrying a drop of DAPI stain over it such that the cells are in direct contact with DAPI.
The DAPI molecules enter the nuclei and bind to the A-T-rich sites of DNA. Now, visualize the cells under a fluorescence microscope. The nuclei appear blue and exhibit distinct morphologies.
The cells showing condensed and fragmented nuclei are indicative of apoptosis, programmed cell death. Others displaying multiple-lobed nuclei represent that cells are experiencing mitotic catastrophe. Those with flattened nuclei showing multiple bright spots are suggestive of cells undergoing senescence.
